摘要
目的:探讨加减薯蓣丸对血管性痴呆(VD)模型大鼠海马髓鞘损伤的影响及其作用机制研究。方法:选取SPF级SD雄性大鼠60只,随机分为假手术组,模型组,加减薯蓣丸高、中、低剂量组,每组12只。除假手术组外,其余4组均采用双侧颈动脉结扎改良法进行VD模型制备,造模结束后通过水迷宫筛选造模成功大鼠进行灌胃。加减薯蓣丸高、中、低剂量组灌胃剂量分别为10、5、2.5 g·kg^(-1)·d^(-1),其余组给予等体积生理盐水。28 d灌胃结束后,通过水迷宫检测大鼠空间学习记忆能力,苏木素-伊红(HE)染色法观察海马区神经元结构,生化检测海马肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、谷氨酸(Glu)含量变化,免疫荧光检测海马髓鞘碱性蛋白(MBP)、活化星形胶质细胞标记物胶质纤维酸性蛋白(GFAP)、半通道蛋白43(Cx43)的表达,电镜观察大鼠海马髓鞘结构,蛋白免疫印迹法(Western blot)检测海马α-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体(AMPAR)、Cx43蛋白表达。结果:与假手术组比较,VD模型大鼠逃避潜伏期显著延长(P<0.01),穿越原平台次数和目标象限滞留时间百分比显著减少(P<0.01);海马CA1区神经元结构紊乱;髓鞘板层疏松,边缘模糊不清;海马CA1区神经元TNF-α、IL-6、Glu含量明显增多,以Glu最为显著(P<0.01);AMPAR蛋白表达显著降低(P<0.01),磷酸化(p)-AMPAR、Cx43蛋白表达显著升高(P<0.01);髓鞘MBP蛋白表达显著降低,GFAP与Cx43共标荧光显著增强。与模型组比较,给予加减薯蓣丸干预后,加减薯蓣丸中药组逃避潜伏期明显缩短(P<0.05),穿越原平台次数和目标象限滞留时间百分比明显延长(P<0.05);海马神经元排列较紧密;髓鞘板层更致密,边缘清晰;海马TNF-α、IL-6、Glu含量明显降低,Glu下降最为显著(P<0.01);AMPAR蛋白表达显著升高(P<0.01),p-AMPAR、Cx43蛋白表达显著降低,以高剂量组最为显著(P<0.01);髓鞘MBP蛋白表达显著增加,GFAP与Cx43共标荧光显著降低、高剂量组改善最为明显。结论:加减薯蓣丸能改善VD大鼠学习记忆能力,其机制可能与抑制Cx43表达,减少Glu外排,抑制AMPAR介导的炎性反应并减少星形胶质细胞标记物GFAP产生,促进髓鞘MBP蛋白表达,缓解髓鞘损伤有关。
Objective:To investigate the effect and mechanism of modified Shuyuwan(SYW)on hippocampal myelin sheath injury in vascular dementia(VD)model rats.Method:Sixty male SD rats of SPF grade were randomly divided into sham operation group,model group,and high-,medium-and low-dose modified SYW groups,with 12 rats in each group.The VD model was induced by bilateral carotid artery ligation in rats of the groups except for those of the sham operation group.After modeling,rats were screened by the water maze test,followed by drug treatment by gavage.Specifically,rats in the modified SYW groups were treated with modified SYW at 10,5,2.5 g·kg^(-1)·d^(-1),accordingly,and those in other groups were administered with the same amount of normal saline.After intragastric administration for 28 days,the spatial learning and memory abilities of rats were detected by the water maze test.The hippocampal neuron structure was observed by hematoxylin-eosin(HE)staining.The content of hippocampal tumor necrosis factor(TNF)-α,interleukin-6(IL-6),and glutamate(Glu)was observed by biochemical detection.The hippocampal expression of myelin basic protein(MBP),astrocyte activation marker glial fibrillary acidic protein(GFAP),and connexin 43(Cx43)was detected by immunofluorescence detection.The myelin sheath structure in the hippocampus was observed by the electron microscope.Theα-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor(AMPAR)and Cx43 protein expression was detected by Western blot.Result:Compared with the sham operation group,the model group showed prolonged escape latency(P<0.01),decreased times of crossing the original platform and percentage of target quadrant detention time(P<0.01),disordered neuron structure in the hippocampal CA1 region,loose myelin sheath lamella with blurry edge,up-regulated expression levels of TNF-α,IL-6,and Glu in the hippocampal CA1 region,especially Glu(P<0.01),reduced expression of AMPAR(P<0.01),increased protein expression of p-AMPAR and Cx43(P<0.01),significantly dwindled protein expression of MBP in the myelin sheath,and enhanced fluorescence co-labeled by GFAP and Cx43.Compared with the model group,the modified SYW groups showed shortened escape latency(P<0.05),increased times of crossing the original platform and percentage of target quadrant detention time(P<0.05),closely arranged hippocampal neuron structure,denser myelin sheath lamella with clear edge,down-regulated expression levels of TNF-α,IL-6,and Glu in the hippocampal CA1 region,especially Glu(P<0.01),up-regulated AMPAR(P<0.01),reduced protein expression of p-AMPAR and Cx43,especially in the high-dose group(P<0.01),significantly elevated protein expression of MBP in the myelin sheath,and weakened fluorescence co-labeled by GFAP and Cx43,especially in the high-dose group.Conclusion:Modified SYW can improve the learning and memory abilities of VD rats,and the mechanism may be related to the inhibition of Cx43 expression,reduction of the release of Glu,inhibition of AMPAR-mediated inflammatory response to reduce the production of astrocyte marker GFAP,and promotion of the expression of MBP protein to alleviate myelin injury.
作者
王建婷
谭子虎
周剑杰
王静
杨琼
刘煜
李贤炜
WANG Jianting;TAN Zihu;ZHOU Jianjie;WANG Jing;YANG Qiong;LIU Yu;LI Xianwei(Hubei University of Chinese Medicine,Wuhan 430065,China;Hubei Provincial Hospital of Traditional Chinese Medicine,Wuhan 430065,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2023年第7期38-46,共9页
Chinese Journal of Experimental Traditional Medical Formulae
基金
湖北省自然科学基金重点项目(2015CFA089)
湖北省中医院老年呆病“五专”项目。
作者简介
第一作者:王建婷,在读博士,从事中医药防治心脑血管疾病的研究,E-mail:Jetty.cmx.ki@gmail.com;通信作者:谭子虎,博士,主任医师,博士生导师,从事中医药防治心脑血管疾病的研究,E-mail:tanzihu2008@163.com。
